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Objectives

Students should be able to:

  1. Describe and apply the process of paper chromatography as it relates to the separation of plant pigments

  2. Describe the role of beta-carotenes, xanthophylls, and chlorophylls a and b in the process of photosynthesis

  3. Describe and apply the process of reduction as it relates to the process of photosynthesis.

  4. Describe, in general, the process of photosynthesis.

  5. Interpret the absorption graph done in Lab Exercise #1 ("Tools" and Measurement) in terms of your understanding of photosynthesis.

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Introduction

The process of photosynthesis is found in plants, some bacteria, and some fungi. Its process is similar in these organisms, but we will study the process as it occurs in plants. Photosynthesis is a biochemical pathway that provides materials for the production of energy during cellular respiration. Plants are able to "capture" the energy provided by sunlight and use it to transform "raw" materials into carbohydrates, principally glucose.

The overall balanced equation for the photosynthetic process is shown below. Do not be misled by its simplicity. The process is very complex.

6CO2  + 6H2O   + Light Energy    C6H12O6 + 6O2

The photosynthetic process takes place in the chloroplasts of the plant cell, specifically on the thylakoid membranes, which are found in the grana. The grana are surrounded by the stroma portion of the chloroplast. The end product, PGAL (phosphoglyceraldehyde), is converted by the cell into glucose or several other chemicals which the cell can utilize.

The process is affected by the light intensity, duration and quality, and how much carbon dioxide, water and oxygen are present. You will investigate these in the lab activities.

The plant pigments, chlorophyll a and b directly capture the energy of the violet / blue (440 - 460 nm) and orange / red (650 - 680 nm) wavelengths of sunlight. The accessory pigments, beta carotene and xanthophyll, capture the energy of blue (450 - 500 nm) wavelength of sunlight. You will be separating these pigments by the chromatography process.

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Pre-lab Activities

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This Laboratory Activity on Photosynthesis can be done virtually as a computer simulation. If you choose to do this lab in the virtual mode, you do not need to do Pre-Lab Activity #1, however, you do need to do Pre-Lab Activity #2. Make sure you answer the Pre-Lab Questions too. You will need to use the following spreadsheet to graph and analyze the data collected from the Light Reactions Lab. You may download it now and save it or you can open it when you are ready to graph your data.

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Light Reactions Spreadsheet

Click here to do this lab as a Computer Simulation.

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Pre-Lab Activity #1

Retrieve your graph of the spectral analysis of chlorophyll and bring it to lab, or you may use this one. If you have access to a color printer this an excellent graph in color.

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Spectral Analysis of Chlorophyll

Download and print out the following:

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Data Collection Sheet for the Study of Photosynthesis Using the Dye Reduction Technique

Download and print out the following:

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Data Collection Sheet for Chromatographic Separation of Chlorophyll Pigments

Download:

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Light Reactions Spreadsheet.

Using the resources available to you find a labeled diagram of a chloroplast. Print it, and bring it to lab.

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Pre-Lab Activity #2

Using Access Science (LRC), find information on the following and bring it to the laboratory session

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Photosynthetic pigments (brief information on the types, their color and at what wavelengths they absorb best)

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Melvin Calvin (biography) Print it out

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Diagram of the internal structure of a dicotyledonous leaf (Print it out)

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Pre-Lab Questions (html version)   Pre-Lab Questions (Word version)

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The Laboratory Activities and Data Collection

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Lab Activity #1 - Chromatographic Separation of Chlorophyll Pigments

Materials:

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Chlorophyll Solution (provided by the instructor)

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Chromatography Materials:

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Chromatography Jar

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Chromatography Paper

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*Chromatography Solvent (ether / acetone 9:1)
 

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Capillary Tubes

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Small plastic ruler

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Pencil

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Data Collection Sheet for Chromatographic Separation of Chlorophyll Pigments
 

* Chemical Hazard - MUST be used with supervision by the instructor and with the fume hood in operation.


Procedures:

  1. Obtain a piece of chromatography paper

  2. On the long edge of the chromatography paper measure up 2 cm. from the bottom and with a pencil place a small dot. Repeat this with the other edge. Draw a faint pencil line connecting the two dots. Click here to see diagram

  3. Using the small capillary tube, withdraw a sample of chlorophyll solution by placing the capillary tube in the solution. It will rise up in the tube by capillary action.

  4. Place your finger over the upper end of the tube and place the lower end of the tube on the dot at the end of the pencil line you have drawn. Slowly release your finger from the upper end of the tube and at the same time draw the tube along the pencil line. Allow the solution to dry.

  5. Repeat the above process 20 times allowing the chlorophyll solution to dry before adding the next application of chlorophyll solution.

  6. Go to the fume hood and select the numbered chromatography jar for your lab group.

  7. Prepare the chromatography paper for the chromatography jar as per instructions from the teacher.

  8. Unscrew the lid of the chromatography jar and carefully place the chromatography paper inside with the bottom of the paper immersed in the chromatography solution. The instructor will have placed about 10 mL of the chromatography solvent in the jar. Gently screw the cap on being careful not to shake the jar.

  9. Let the process run until the chromatography solution is approximately 1 cm from the top of the chromatography paper. (Check on this every 3 minutes)

  10. Remove the chromatography paper from the jar, and let it dry under the fume hood.

  11. Analyze the results (See Data Collection Sheet for this Lab Activity)

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Lab Activity #2 - The Study of Photosynthesis Using the Dye Reduction Technique

Materials:

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See Handout

Procedure:

  1. Follow the procedures set forth in the handout

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Post-lab Activity and Data Analysis

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Results and Analysis:

  1. Graph the data from the dye reduction activity and analyze the results.

  2. Analyze the Chromatographic Separation of Chlorophyll Pigments. If you did this lab as a computer simulation you have completed Item #2. Attach them to your Post-Lab Questions

  3. Answer the Post-Lab Questions

  4. Scan, print, and attach the Chromatograph for your lab group.

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Post-Lab Questions (html version)     Post-Lab Questions (Word version)

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